Weather damage or preharvest sprouting in wheat, is caused by the action of hydrolytic enzymes (amylases, proteases and lipases) in the grain endosperm. These enzymes (triggered by rain at or just before harvest), accelerate the breakdown of starch granules and protein in the endosperm of germinating grain (Meredith, P.; Pomeranz, Y. Advances in Cereal Science and Technology 7 (1985) 239-299). Wheat that is weather-damaged has a significantly lower market value as a result of being rendered unsuitable for human consumption. This is because the products that are made from sprouted wheat, for example breads, have grey colour, crumb texture, loaf structure and volume or in the case of noodles, poor colour and cooking qualities, due to the action of these hydrolytic enzymes, which include alpha-amylases (Orth, R. A.; Moss, H. J. Proceedings of the Fourth International Conference on Pre-harvest Sprouting, D. Mares (Ed.) Westview Press, Boulder, Colo., USA (1987) 167-175; Derera, N. F. (Ed.) Preharvest sprouting in cereals, CRC Press Inc., Boca Raton, Fla., USA (1989)).
At grain delivery to the silo or elevator or during harvesting, mixing of a small quantity of damaged grain with larger amounts of sound grain can lead to downgrading of all of the grain. The necessity for accurately discriminating sprouted from sound wheat highlights the need for a quick, easy and reliable test for preharvest sprouting. The most common method for detecting preharvest sprouting at elevators involves the measurement of alpha-amylase activity using the “Falling Number” method, in which the consequences of enzymic hydrolysis of starch caused by amylase production is assessed as the time required for a plunger to fall through a heated slurry of wholemeal and water (Hagberg, S. Cereal Chemistry 37 (1960) 218; Perten, H. Cereal Chemistry 41 (1964) 127-140). However, the capital cost of the instrument means that it is only feasible to install them at a limited number of mills or major grain silos or elevators. The method is also relatively low in throughput and results can be affected by variation in starch pasting characteristics (D'Appolonia, B. L.; Macarthur, L. A., Pisesookbuntererng, W.; Ciacco, C. F. Cereal Chemistry 59 (1982) 254-257; Ringlund, K. Proceedings of the Third International Symposium on Pre-Harvest Sprouting in Cereals, J. E. Kruger and D. E. LaBerge (Eds.) Westview Press, Boulder, Colo., USA, (1983) 111-118).
The cheaper option of visual assessment is both unreliable and not objective (Jensen, S. A.; Munck, L.; Kruger, J. E. Journal of Cereal Science 2(1984) 187-201), while other methods such as the Rapid ViscoAnalyzer (Ross, A. S.; Orth, R. A.; Wrigley, C. W. Proceedings of the Fourth International Symposium on Pre-Harvest Sprouting in Cereals, D. J. Mares (Ed.) Westview Press, Boulder, Colo., USA (1987) 577-583) and Near Infrared analysis (Czuchajowska, Z.; Pomeranz, Y. Preharvest Sprouting in Cereals 1992, M. K. Walker-Simmons and J. L. Ried (Eds.) American Association of Cereal Chemists, St Paul, Minn., USA (1992) 409-416), although faster, involve high capital cost. Near Infrared predictions of Falling Number are also of relatively low precision and can only discriminate relatively large differences in Falling Number (Osborne, B. G. Journal of the Science of Food and Agriculture, 35 (1984) 106-110; Czuchajowska, Z.; Pomeranz, Y.; Preharvest Sprouting in Cereals 1992, M. K. Walker-Simmons and J. L. Ried (Eds.) American Association of Cereal Chemists, St Paul, Minn., USA (1992) 409-416; Shashikumar, K.; Hazleton, J. L.; Ryu, G. H.; Walker, C. E. Cereal Foods World 38 (1993) 264-269). Direct enzyme activity assays for alpha-amylase (Barnes, W. C.; Blakeney, A. B. Die Starke 6 (1974) 193-197; McCleary, B. V.; Sheehan, H. Journal of Cereal Science 6 (1987) 237-251) are not suited for silo (elevator) or on-farm use due to a need for technical expertise and equipment such as waterbaths and filtration devices.
Immunoassays provide alternative methods for detection of preharvest sprouting through the use of antibodies that are specific for alpha-amylase isozymes. Alpha-amylases are considered to be the most appropriate targets for a test because: 1. they are relatively abundant, 2. they are synthesized early in the preharvest sprouting sequence (Corder, A. M., and Henry, R. J. Cereal Chemistry 66 (1989) 435-439), 3. they are responsible for many of the quality defects that occur when end products are prepared from sprouted wheat, and 4. the basis of the measurements in the “industry standard test” (Falling Number) is changes in the viscosity of a wholemeal-water slurry due to the presence of carbohydrate-degrading enzymes such as amylases. Earlier research has shown that specific antisera can be developed for separate recognition of the two major groups of alpha-amylase isozymes (Daussant, J.; Renard; H. A. Cereal Research Communications 4 (1976) 201-212; Lazarus, C. M.; Baulcombe, D. C.; Martienssen, R. A. Plant Molecular Biology 5 (1985) 13-24). Immunoassay techniques have an added potential advantage over enzyme activity assays, in that by using appropriate amylase antibodies it should be possible to specifically measure different amylase isozyme families. Immunoassay kits are generally quite robust and suitable for shipping and use in harsh environments, and can be used by individuals with little training. Such tests could not only be used by grain handlers or traders at silo (elevator) delivery of grain, but also by individual wheatgrowers. This would allow them to detect sprouting on-farm prior to harvesting in order to prevent contamination of sound wheat by sprouted grain.
The most sensitive, specific and quantitative immunoassays require the use of both a solid-phase bound antibody and a labelled detection antibody in a “two-site” assay. The detection antibody may be labelled with an enzyme, coloured particle or sol, or radioactive element or fluorophore. However, for field use without special equipment, the most useful methods are those in which the test result can be interpreted visually. The present invention relates to the development of two-site immunoassays for the qualitative or quantitative detection of alpha-amylase.